29 March 2013. Reducing activity in the thalamus of mice can give rise to cognitive impairments similar to those seen in schizophrenia, reports a study that came out March 21 in Neuron. Led by Christoph Kellendonk of Columbia University, New York, the study paired a designer receptor with a designer drug to selectively quiet neuronal activity in the mediodorsal (MD) thalamus, a region with dense, two-way connections with the prefrontal cortex. This manipulation weakened synchronous activity between MD thalamus and prefrontal cortex, and triggered impairments in cognitive flexibility and working memory reminiscent of those found in schizophrenia.

Click image to see a mini-lecture on this paper at the Cell Press YouTube channel.

The findings argue that problems with the thalamus may be sufficient to induce some of the cognitive deficits encountered in schizophrenia. When it comes to sophisticated mental processes like working memory, the thalamus does not figure as prominently as prefrontal cortex does, but its connections with brain regions far and wide makes it plausibly influential. In schizophrenia, brain imaging has turned up abnormalities in the thalamus (see SRF related news story and SRF news story), including reduced activity in the mediodorsal nucleus of the thalamus (Minzenberg et al., 2009), echoing earlier evidence of structural abnormalities in this region (reviewed in Alelú-Paz and Giménez-Amaya, 2008. But it’s hard to know whether these reflect a cause or consequence of schizophrenia symptoms.

To address this question, the researchers modeled the decrease in mediodorsal nucleus activity in mice using the “designer-receptor exclusively activated by a designer drug” (DREADD) system developed by coauthor Bryan Roth (Armbruster et al., 2007). In this system, a receptor is engineered and introduced to specific regions of the brain, then activated at will with a drug. Specifically, the researchers introduced a mutated human muscarinic receptor (hM4D) into the MD thalamus. This receptor was no longer activated by its usual ligand, acetylcholine, or to any other neurotransmitters; instead, its ligand was clozapine-N-oxide (CNO), an otherwise inert chemical. When activated, the hM4D receptors hyperpolarized neurons by turning on potassium channels. Thus, when the researchers wanted to tone down MD thalamus activity, they injected mice with CNO. Unlike a lesion, this provided a graded manipulation of activity.

Flexibility and memory
First authors Sebastien Parnaudeau and Pia-Kelsey O’Neill began by verifying that their DREADD system worked as planned. The hM4D receptor was expressed exclusively in neurons, including those projecting to the prefrontal cortex. Within minutes, a CNO injection suppressed firing in about half the neurons they recorded. The neurons did not go silent, however, but rather decreased their firing rate by an average of 38.7 percent. These changes were not observed when control mice without the hM4D receptor were given CNO.

The researchers then tested the effects of turning down MD thalamus activity on reversal learning, a measure of cognitive flexibility, which is impaired in schizophrenia. In this paradigm, choosing one stimulus earns a reward, and choosing another gives a penalty. After this pairing is learned, the rule changes so that choosing the stimulus previously associated with a reward gives a penalty, and vice versa. With CNO on board, the hM4D mice could learn the first set of pairings as well as did controls, which included three groups: mice without the hM4D receptor given CNO, mice without the hM4D receptor given saline, and mice with the hM4D receptor given saline. When the pairings were reversed, however, the hM4D receptor-CNO mice didn’t adapt as well as the control groups did, and continued to choose the stimulus previously associated with reward.

To probe working memory, also affected in schizophrenia, the researchers tested the mice in a T-maze task. There, the mice explored the maze to discover which of two arms held a reward; after a delay, their memory for this arm was tested by having them choose the arm that did not originally contain the reward. With CNO on board, the hM4D receptor mice struggled to learn the task, taking about two days longer to reach the same level of correct choices attained by the three control groups. CNO also impaired how well a separate group of mice already trained on the task could show what they remembered: hM4D receptor mice chose the correct arm less often than the control group when given CNO. This suggests that decreased MD thalamus activity mucked with working memory, or the ability to keep information online. Other tests indicated that this manipulation did not interfere with attention, locomotion, and the ability to use spatial information.

Trouble in the beta band
Quieting activity in MD thalamus may have interfered with cognitive flexibility and working memory by disrupting its communication with prefrontal cortex, a region crucial for these mental processes. The researchers assessed the state of this connection by measuring the synchronous activity between MD thalamus and the medial prefrontal cortex (mPFC). As mice performed the T-maze working memory task on which they had already been trained, the researchers found enhanced synchrony in the beta-frequency range, which consists of oscillations between 13-20 Hz, but not in gamma or theta frequencies. Upon treating hM4D mice with CNO, however, this beta-band synchrony weakened, meaning that the MD thalamus and mPFC were often out of step with each other. The decrement in synchrony came with a drop in percentage of correct choices mice made during T-maze performance. For both saline- and CNO-treated animals, a robust correlation between beta-band synchrony strength and accurate choices emerged, which suggests that the pattern of activity flowing between MD thalamus and prefrontal cortex is important for retaining information.

Though beta oscillations are not as well known as theta or gamma oscillations, they, too, have links to cognition, and have been reported as impaired in schizophrenia (Uhlhaas et al., 2006). Although the disrupted beta oscillations and cognitive deficits originated from a subtle manipulation of MD thalamus in this paradigm, it remains to be seen whether the decrease in MD nucleus activity found in schizophrenia reflects a core problem of this region, or is a downstream consequence of a problem elsewhere in the brain. Still, the study suggests that a graded manipulation of a single region can be a fruitful way to explore isolated parts of circuits and their contributions to higher mental processes.—Michele Solis.

Schizophrenia and Thalamus Dysfunction
The pathophysiology of schizophrenia still conceals many secrets (Meyer-Lindenberg, 2010; Insel, 2010), which is the reason why the neurobiology of schizophrenia is the relentless object of experimental and clinical investigations. These point out that schizophrenia is a complex, multifunctional disorder involving dysfunctional networks.

Guller and colleagues recently published, in the Archives of General Psychiatry (published online March 5, 2012), clinical data in favor of the hypothesis of thalamic dysfunction in schizophrenia. The authors’ objective was to test the hypothesis that the thalamus of patients with schizophrenia responds abnormally to cortical activation. The authors used fMRI to record in healthy subjects and patients with schizophrenia the vascular response in their cerebral cortex (medial superior frontal cortex [mSFC] and insula) and thalamus following a single pulse TMS (spTMS) of the precentral gyrus. The authors recorded a reduced hemodynamic response in the thalamus, mSFC, and insula in the patients. Taken together, these results reveal abnormal decreases in thalamus-mSFC and thalamus-insula functional connectivity. The authors conclude that the thalamus contributes to the patterns of aberrant connectivity, a central feature of schizophrenia. This interesting study opens up new vistas, leaving a certain number of questions open.

What are the corticothalamic (CT)-induced neural mechanisms underlying the thalamic hemodynamic response?
No definitive answer can be given since the TMS-induced effects depend on many factors, including TMS protocol, the cortical target, and state, as previously demonstrated by the same research team (Massimini et al., 2007; 2010). The subjects of the study by Guller et al. were awake with open eyes. During wakefulness, TMS evokes a time-locked high-frequency (20-35 Hz) sustained EEG oscillation during the first 100 ms (Massimini et al., 2010). Such a cortically evoked, short-lasting oscillation is expected to reflect simultaneous waves of excitation and inhibition in the CT/thalamocortical (TC) systems. On the other hand, the neural mechanisms underlying the slowness of the BOLD neuro-glio-vascular response remain elusive.

Let us consider the appealing concept of “grouping of brain rhythms in CT systems” (Steriade, 2006). Indeed, the grouping of brain rhythms in CT/TC systems is the result of interplays between synaptic and intrinsic properties of the corresponding neural excitatory and inhibitory elements. Such functional-anatomic interactions would thus generate state-dependent complex wave sequences, which are readily apparent in spontaneously occurring cortical EEG. Therefore, it is tempting to suggest that every spTMS evoked both excitation and inhibition in the target cortical area with their respective amount and time course, which would subsequently modulate the activity of local and distant structures, including the thalamus potentially inducing the BOLD response. The CT-induced activity pattern depends on the anatomical and electrophysiological properties of the engaged nerve elements. Among the most responsive thalamic elements to CT activation are the GABAergic thalamic reticular nucleus (TRN) neurons (Pinault, 2004). TRN cells can behave like resonators. For instance, in the awake rat, it was demonstrated that, during natural CT 5-9 Hz oscillations, TRN cells start to fire with robust, high-frequency bursts of action potentials in a rhythmic manner and almost always do so before TC neurons (Pinault, 2003). It is worth specifying that the intracellular events underlying CT 5-9 Hz and TC spindle oscillations are distinctively different in thalamic neurons (Pinault et al., 2006). The rodent CT 5-9 Hz oscillation emerges during quiet, immobile wakefulness and is thought to be a sensorimotor rhythm, and it may be the equivalent of the alpha rhythm of the human occipital cortex.

What are the mechanisms underlying the smaller thalamic hemodynamic response in patients with schizophrenia?
Guller and colleagues think that the structural abnormalities observed in the thalamus of patients with schizophrenia and the functional-anatomical properties of the thalamo-reticular (TC-TRN) system account for its dysfunction, reflected in their results by the smaller spTMS-induced vascular response (Guller et al., 2012) and by a deficit in sleep spindles (Ferrarelli et al., 2007; 2010). Moreover, structural, metabolic, and neurochemical changes (Clinton and Meador-Woodruff, 2004; Harms et al., 2007; Popken et al., 2000) and abnormalities in the expression of glutamate receptors (Ibrahim et al., 2000) were observed in the brains of patients with schizophrenia.

Assuming that the functional-anatomical properties of thalamic circuits are severely affected in schizophrenia, the impact would be major. Indeed, one cannot think about the thalamus without thinking about the cerebral cortex and vice versa, since they are reciprocally connected and work in tandem to generate physiological and pathological rhythms (Steriade and Deschenes, 1984; Steriade and Llinas, 1988). Both the neocortex and the thalamus and their related CT/TC circuits are prominently involved in the same global brain operations (consciousness, perception, and cognition). The thalamus also innervates the striatum, amygdala, and hippocampus (Lisman et al., 2010). Postmortem and high-resolution functional-anatomical studies support the hypothesis of dysfunctional CT/TC and basal ganglia networks in patients with schizophrenia (Cronenwett and Csernansky, 2010; Clinton and Meador-Woodruff, 2004).

The thalamus relays sensorimotor and higher-order information to the cerebral cortex and plays a key role in large-scale cortico-cortical communication (Guillery and Sherman, 2002). The thalamus is reciprocally connected not only with the cerebral cortex, but also with the TRN. The TC and CT neurons are glutamatergic and cross the TRN, wherein they give off axon collaterals (Jones, 2007). The TRN contains only GABAergic neurons, which project to the dorsal thalamus (Pinault, 2004). The firing pattern (single-action potential or high-frequency burst of APs) of TC and TRN neurons is state and voltage dependent. Pathological changes in the firing pattern of thalamic neurons might involve, among many other factors (Pinault, 2011), potassium channel dysfunction (Vukadinovic and Rosenzweig, 2012).

The TRN is a thin GABAergic layer interface strategically located between the thalamus and the neocortex. It is more than the mediator of selective attention (Pinault, 2004). It works like a combinatorial matrix since it holds and can combine all functional, sensorimotor, limbic, and cognitive modalities. Furthermore, it is endowed with extraordinary intrinsic oscillatory properties. TRN cells work like integrators under the leading influence of CT inputs. Of importance, although the thalamus and the TRN are reciprocally connected, this anatomical rule does not apply at the cellular level. Indeed, TRN and TC neurons principally form two-neuron, open-loop circuits, meaning that they do not form reciprocal connections (Pinault and Deschenes, 1998). The functioning principle of such open GABAergic-glutamatergic circuits is lateral inhibition, which allows the relaying of relevant streams of information to the neocortex and the deletion of distracting activities during brain operations. Disruption of thalamic lateral inhibition is thought to contribute to a lack of coordination between neuronal assemblies, as observed in schizophrenia (Pinault, 2011).

Is the amplitude of the thalamic neuro-glio-vascular response a signature of clinical symptoms?
Guller and colleagues emphasize that the observed changes in sleep spindles (Ferrarelli et al., 2010) and in the amplitude of the thalamic hemodynamic response (Guller et al., 2012) may be related to clinical symptoms, in particular, to positive symptoms. Both the thalamus and the neocortex work under the influence of neuromodulatory inputs from the forebrain and brainstem. Disorders in neuromodulatory transmission might generate excessive TC background activity, subserving, for instance, the emergence of hallucinations (Behrendt, 2006) and impairing information processing in CT/TC circuits. So, one may wonder whether or not schizophrenia-related thalamic anatomo-functional abnormalities are secondary to dysfunctional pre-thalamic (e.g., neuromodulatory) inputs.

Multi-target drug discovery has typically been neglected in the world of genetics and high-throughput screening because of the difficulty of rationally defining a pharmacological profile, but it has major advantages for treating complex disorders such as schizophrenia. It is no wonder that the currently approved antipsychotics do have a rich pharmacology and substantially improve the clinical phenotype. With so many different genotypes defining individual patients, focusing on only one target is likely to have small effects that might disappear in clinical trials with larger patient populations. Even over all indications (not only CNS), more than half of the first-in-class medicines approved in the last decade have been found by using phenotypic assays and have typically multi-target pharmacology (Swinney and Anthony, 2011).

The approach presented here suggests a rational way to identify 1) a set of targets and 2) chemical structures that might serve as hits for further medical chemistry development. It might therefore alleviate the concerns of many medical chemistry departments in pharmaceutical companies.

Changing the mindset from developing the next extremely specific and potent inhibitor to pursuing multi-target pharmacology is urgently needed to break the deadlock of unsuccessful new drug development in schizophrenia.